B8
An optical proteomic profile of the ErbB family in solid tumours exposed to tyrosine kinase inhibitors (TKIs)
Gargi Patel1, Muireann Kelleher1, Luis Fernandes1, Franca Fraternali1, Enyi Ofo1, Gilbert Fruhwirth1, Cheryl Gillett1, James Spicer1, Emma McLean2, George Santis1, Paul Cane2, Paul Barber3, Boris Vojnovic3, Paul Ellis2, Tony Ng1
1Kings College London, UK, 2Guys & St Thomas' NHS Trust, London, UK, 3Gray Institute for Radiation Oncology & Biology, University of Oxford, UK
Background
This project aims to develop molecular diagnostic assays for ErbB TKIs to identify tumours with quantifiable changes in ErbB protein complex formation and trafficking in response to therapies. Fluorescence lifetime imaging microscopy (FLIM) was used to quantify protein-protein interactions by measurement of Frster resonance energy transfer (FRET), which takes place between two interacting partners in close proximity (nanometer scale). This novel technology is used to demonstrate ErbB2/3 dimerisation and ErbB2 ubiquitination, and the effect of perturbation of this system by lapatinib.
Method
MCF-7 breast cancer cells were transfected with fluorophore-conjugated plasmids encoding ErbB2 and ErbB3, or ubiquitin. Energy transfer (FRET) occurs only where these proteins are interacting with one another thus quantifying receptor heterodimerisation or ubiquitination. The effects of stimulation by neuregulin and inhibition by lapatinib on these interactions were studied. Assays for measuring endogenous ErbB heterodimerisation and ubiquitination are being developed. In order to develop a predictive assay these will be applied to cytospin samples from tumour aspirates from an ongoing phase II clinical trial, before and after treatment with an ErbB2 inhibitor.
Results
Formation of the ErbB2/3 heterodimers and ErbB2 ubiquitination are modulated by short term inhibition with lapatinib in a complex manner. Internalisation of the ErbB2/3 dimers is visualised upon inhibitor treatment. The determinants for these TKI effects are being investigated by modelling the molecular dynamics.
Conclusion
This study has provided the initial clues for establishing the multi-parameter dataset required to predict response to TKI treatment of individual tumours at a systems biology level. This technology will be translated to tumour samples in order to develop molecular diagnostic assays which may help to predict sensitivity to TKIs and identify a population of patients most likely to benefit from treatment.
